P070 A novel NGS workflow reveals MICA ALLELE frequencies based on 350,000 German samples

Aim The MICA and MICB molecules serve as ligands of the activating NKG2D receptor expressed on natural killer (NK) cells. Recent studies indicate effects of MIC genotypes on hematopoietic stem cell transplantation (HSCT) outcome. To provide MIC allele information for donor selection, we developed an NGS-based high-throughput genotyping workflow for MICA and MICB and applied it for genotyping registry donor samples. Methods Exons 2 and 3, as well as most of exons 4 and 5 of MICA and MICB are amplified in a multiplexed PCR reaction. The PCR products are sequenced on Illumina HiSeq or MiSeq instruments. The data are processed by an updated neXtype software version to provide allele-level genotyping information. Results Using this NGS based workflow, we genotyped 350,000 donors registered in Germany and report on the observed MICA allele frequencies. Due to the restricted sequence coverage, 9 alleles encoding distinct proteins cannot be resolved. However, the unprecedented depth of the study allowed us to estimate allele frequencies for 49 of the 84 described MICA alleles distinguished at the protein coding level. In addition we identified novel alleles in 0.2% of the samples. The 13 (31) most abundant alleles account for a cumulative allele frequency of 99% (99.99%). Conclusions This newly developed NGS-based genotyping approach offers the opportunity to analyze the genetic diversity of MICA and MICB in large cohorts at high-resolution.